636 research outputs found
Constant codimension fixed sets of commuting involutions
No full textThe ideals
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\mathcal {J}_{ \ast ,k}^r
of cobordism classes in
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Leo Tolstoj-Shakers correspondence (Korespondencja Lwa Tołstoja z shakerami)
No full textThis is an exchange of letters between Leo Tolstoj and the Shakers, American communitarian religious groups. I transcribed them from original manuscripts or microfilms during my research at the Shaker library, Sabbathday Lake, Maine, USA in 2009. The main themes are Christian ethics, pacifism, non-violence etc., reflecting Tolstoj's interpretation of Christianity and the group's communal experience.Jest to, niepełny, zapis korespondencji między Lwem Tołstojem a shakerami, amerykańską wspólnotową grupą chrześcijańską. Transkrypcji z oryginalnych manuskryptów lub mikrofilmów dokonałem podczas pobytu na kwerendzie w Shaker LIbrary w Sabbathday Lake, Maine, USA w 2009 r. Główne tematy listów to etyka chrześcijańska, pacyfizm, niestosowanie przemocy itd., co odzwierciedla, z jednej strony, radykalną tołstojowską interpretację chrześcijaństwa, z drugiej zaś doświadczenia wspólnotowego życia shakerów.This research was financed by the Foundation for Polish Science, within the "Kwerenda" program. (Badania te zostały sfinansowane przez Fundację Nauki Polskiej w ramach programu "Kwerenda"
Molecular mechanisms of the coupling of gating to voltage sensing in transmembrane proteins
No full textVoltage gated potassium ion (Kv) channels regulate action potentials of the nervous system by responding to changes in transmembrane voltage, enabling K+ transport across the membrane to restore cells to their resting potential. Comprised of four identical subunits, Kv channels contain four voltage sensing domains arranged on the periphery of a central pore domain. Each voltage sensor is comprised of four transmembrane helices, numbered S1 through S4. The S4 helix, containing four to six highly-conserved, positively-charged arginine or lysine residues, is responsible for voltage sensitivity in Kv channels. The pore domain consists of two transmembrane helices, S5 and S6. The S5 helix constitutes the periphery of the pore domain and is believed to be relatively immobile. The S6 helices, lining the interior of the channel, gate the protein and regulate K+ permeation. Because each subunit of Kv channels contains six transmembrane helices, they are often referred to as 6TM Kv channels.
The depolarization of an action potential is initiated as sodium ions enter the cell. At the cellular resting potential of -70 mV, potassium ion channels are closed, and the S4 helix is in its “down” state. As the electrochemical gradient changes, the S4 helices of Kv channels begin to reorient within the membrane. At the peak of the action potential (roughly +20 mV), the S4 helices exist in their “up” state. This conformational transition of the S4 helix is coupled to the pore domain via the S4-S5 linker, a short, amphipathic helix along the intracellular membrane-water interface. By bridging the C-terminus of the voltage sensor to the N-terminus of the pore domain, the S4-S5 linker couples the voltage sensitivity of the voltage sensor to K+ conduction in the pore domain.
Because they begin opening at voltages less than 0 mV, all crystal structures of Kv channels contain an open pore domain. With no structure in the closed conformation, the mechanism of gating in Kv channels remains unclear. Nevertheless, significant biophysical studies have revealed insights into both the closed conformation and the gating transition itself. In this dissertation, I will explore questions relevant to the gating mechanism in voltage gated potassium ion channels through fully-atomistic molecular dynamics (MD) simulations.
First, in Chapter 2, I will address the potential role of the 310 helical conformation found in the C-terminal end of S4 in the crystal structures of Kv channels. Spanning eight or more residues, these 310 helices are both uncharacteristically long and conserved in K+ channel crystal structures. By simulating the Kv1.2/2.1 chimera channel’s voltage sensor embedded in a lipid bilayer, I find that an alpha to 310 helical interconversion of the S4 helix reproduces many experimental measurements of the open and closed states of Kv channels.
In Chapter 3, I perform molecular dynamics simulations of the entire Kv1.2/2.1 chimera channel. First, I examine the impact of an alpha to 310 helical interconversion of the S4 helix on the pore domain of the channel. Though the results are consistent with the results in Chapter 2 (and the corresponding experimental measurements), I find that this secondary structural modification is insufficient to influence the pore domain of the channel on the timescale of my simulations. In the second half of Chapter 3, I use molecular dynamics simulations to generate a closed state model of the Kv1.2/2.1 chimera from luminescence resonance energy transfer (LRET) measurements of the closed conformation of KvAP. The resulting structure is indeed closed, and also recapitulates a number of experimentally determined measurements of the closed channel.
In Chapter 4, I focus on the pore domain. First, using targeted molecular dynamics simulations, I generate a transition between a closed model of the KvAP linker and pore domain to the open conformation. Then, using an umbrella sampling method, I quantify the energetics of the gating transition in KvAP and assess the physiological implications. In agreement with experimental studies of Kv channel energetics, I find that the open pore is roughly 2.7 kcal/mol lower in free energy than the closed conformation. The targeted molecular dynamics and umbrella sampling simulations reveal additional insights into the gating mechanism of KvAP.
Lastly, in Chapter 5, I use MD simulations to gain insights into the binding mechanism of VSTx1, a Kv channel inhibitor. By using the experimentally determined neutron scattering density profile of the VSTx1 toxin bound to a lipid bilayer as a restraint for molecular dynamics simulations, I recreate the experimental scattering density profile, and also offer insight into the binding of VSTx1 to a lipid membrane
Investigations on subunit-specific assembly and structure-function studies of the voltage sensor in KCNQ potassium channels
No full textA detailed understanding of how potassium channels function is crucial e. g. for the development of drugs, which could lead to novel therapeutic concepts for diseases ranging from diabetes to cardiac abnormalities. An improved understanding of channel structure may allow researchers to design medication that can restore proper function of these channels. This is particularly important for KCNQ channels, since four out of five family members are involved in human inherited disease. In addition to structure and function relationships the determinants which govern assembly of KCNQ subunits are decisive to understand the physiological role of the KCNQ channel family members. Many details of KCNQ channel assembly remain incompletely understood. Previous work has shown that the subunit-specific heteromerisation between KCNQ subunits is determined by a ~115 amino acid-long subunit interaction domain (si) within the C-terminus (Schwake et al., 2003). Recently, Jenke et al. (2003) proposed that the C-terminal domains in eag and erg K+ channels act as sites which drive tetramerization. From their ability to form coiled coils, these domains were referred to as tetramerizing coiled-coil (TCC) sequences. Jenke et al. also pointed out that KCNQ channels contain bipartite TCC motifs within their C-termini, exactly within the si domain, which is responsible for the subunit-specific interaction pattern. The first part of this thesis was dedicated to determine the individual role of these TCC domains on homomeric and heteromeric channel formation in order to further characterize the molecular determinants of KCNQ channel assembly. In the second part of this thesis cystein-scanning mutagenesis was employed, followed by thiol-specific modification using MTS reagents to screen more than 20 residues in the S3-S4 linker region and in the S4 transmembrane domain of the KCNQ1 channel to gain information about residue accessibility, the functional effects of thiol-modifying reagents (MTSES), and effects of crosslinking selected pairs of Cys residues by Cd+ ions, which could be used for testing model predictions based upon known Kv channel structures from the literature. According to homology modelling based on the Kv1.2 structure it was attempted to determine the proximity of individual residues from different transmembrane segments using the metal bridge approach (crosslinking by Cd+ ions). This led us to derive structural constraints for interactions between the S4 voltage sensor and adjacent transmembrane segments of KCNQ1. Similar studies have previously been performed on the Shaker K+ channel, which has served as a paradigm for structure-function research of voltage-gated K+ channels for a long time, but little is known for KCNQ channels concerning their similarity to published K+ channel structures.Die Fähigkeit von KCNQ-(Kv7)-Kanälen Hetero-Oligomere aus verschiedenen KCNQ-Untereinheiten zu bilden, ist von hoher physiologischer Bedeutung, da Heteromere von KCNQ3 mit KCNQ2 oder KCNQ5 dem neuronalen M-Strom zugrunde liegen, welcher die neuronale Erregbarkeit moduliert. Die fünf Mitglieder dieser Familie (KCNQ1-KCNQ5) sind homolog zu Shaker Kanälen jedoch unterscheiden sie sich in ihrer Struktur dadurch, daß die KCNQ-Kanäle längere Carboxytermini haben, die intrazellulär lokalisiert sind. Außerdem führen Mutationen bei vier von den fünf Mitgliedern dieser Familie (KCNQ1-KCNQ4) zu genetischen Störungen. KCNQ1-Mutationen verursachen das Romano-Ward-Syndrom (RWS), eine Sonderform (Typ1) des Long-QT-Syndroms (LQT). Dieser Name leitet sich von der charakteristischen Verlängerung des QT–Intervalls im Elektrokardiogramm ab. Dabei handelt sich um eine dominant vererbliche Repolarisations-Störung des Herzmuskels. Diese kann Ursache für Herzrhythmusstörungen sein, die häufig zu plötzlichen Herztod führen. Bestimmte rezessive KCNQ1- und KCNE1-Mutationen sind zudem für das Jervell-und-Lange-Nielsen-Syndrom verantwortlich. Bei dieser Mutation leiden die Patienten zusätzlich zu kardialen Abnormalitäten auch noch unter progressiver Taubheit. KCNQ2- und KCNQ3-Mutationen verursachen die dominant vererbliche Form der Neugeborenenepilepsie „benign famillial neonatal convulsions“ (BFNC). Die molekulargenetische Ursache dieser Erkrankung ist ein KCNQ2/KCNQ3-Heteromer. Diese K+-Kanal-Untereinheiten werden hauptsächlich im Gehirn exprimiert und repräsentieren das molekulare Korrelat des sogenannten M-Stroms, welcher über muskarinerge Stimulation (M1-Rezeptoren) reguliert wird. KCNQ4-Mutationen können eine dominant vererbliche Taubheitsform, DFNA2 genannt, verursachen. Der KCNQ5-Kanal weist ein breites Expressionsmuster auf, für den aber noch keine Beteiligung an menschlichen Erbkrankheiten nachgewiesen werden konnte. Innerhalb dieser Familie unterscheiden sich KCNQ1 und KCNQ3 am meisten hinsichtlich ihrer Eigenschaft, Heteromere mit anderen KCNQ-Untereinheiten zu bilden. Im C-Terminus von KCNQ-Kanälen wurde vor kurzem eine ca. 100 Aminosäuren umfassende Domäne identifiziert, welche die Spezifität des Untereinheiten-Zusammenbaus bestimmt. Innerhalb dieser sog. si-Domäne gibt es zwei Abschnitte, die jeweils aus ~30 Aminosäuren bestehen und eine hohe Wahrscheinlichkeit zur Ausbildung einer coiled-coil-Struktur aufweisen. Durch Übertragung der ersten oder der zweiten coiled-coil- bzw. TCC-Domäne von KCNQ3 auf KCNQ1 wurden KCNQ1(TCC1)Q3- und KCNQ1(TCC2)Q3-Chimären generiert, die sich beide mit KCNQ2 koimmupräzipitieren ließen. Allerdings konnte nur mit KCNQ1(TCC2)Q3 eine Erhöhung der Ströme und der Oberflächenexpression in Koexpression mit KCNQ2 beobachtet werden, wie sie für die KCNQ2/KCNQ3-Interaktion charakteristisch ist. Die Deletion von TCC2 innerhalb von KCNQ2 resultierte zwar in funktionalen homomeren Kanälen, es entfiel jedoch die Zunahme des Stromes nach der Koexpression des Deletionskonstrukts mit KCNQ3. Im Gegensatz dazu konnten nach Deletion von TCC1 innerhalb von KCNQ2 keine funktionalen homomeren KCNQ2- bzw. heteromere KCNQ2/KCNQ3-Kanäle beobachtet werden. Auch hoben Mutationen, die die vorhergesagte coiled-coil-Struktur von TCC1 in KCNQ2 oder KCNQ3 unterbrachen, die funktionelle Expression dieser Konstrukte einzeln oder in Kombination mit der jeweils intakten anderen Untereinheit auf. Demgegenüber waren homomere KCNQ2-Kanäle mit helix-brechenden Mutationen in TCC2 zwar funktionell, es unterblieb jedoch die Heteromerisation mit KCNQ3. Im Gegensatz dazu hetero-oligomerisierte ein KCNQ3-Konstrukt mit einer coiled-coil-brechende Mutation in TCC2 weiterhin mit KCNQ2. Die Daten in dieser Arbeit unterstreichen, dass die TCC1-Domänen von KCNQ2 und KCNQ3 notwendig sind, funktionale homomere als auch heteromere Kanäle zu bilden, während beide TCC2-Domänen für einen effizienten Transport heteromerer KCNQ2/KCNQ3-Kanälen zur Plasmamembran verantwortlich sind. Die funktionellen Eingenschaften von KCNQ-Kanälen unterscheiden sich in vielerlei Hinsicht deutlich von z. B. denen von Shaker K+-Kanälen aus Drosophila , welche als am besten untersuchte Kv-Kanäle betrachtet werden können. Um einen systematischen Beitrag zu Struktur-Funktions-Beziehungen an KCNQ-Kanälen zu erbringen, wurde im zweiten Teils dieser Arbeit der Versuch unternommen, den Einfluss verschiedener Aminosäuren-Seitenketten durch eine Cystein-Scanning-Mutagenese im Bereich des S4-Segments und der vorangehenden S3-S4-Schleife von KCNQ1 zu analysieren. Um die extrazelluläre Zugänglichkeit der eingeführten Cysteine zu detektieren, wurde nach Effekten extrazellulärer Applikation der thiol-spezifisch reagierenden Substanz MTSES auf die funktionellen Eigenschaften der Kanäle gesucht. Alle 20 untersuchten Cystein-Mutanten konnten in Xenopous Oozyten funktionsgemäß exprimiert werden, mit Ausnahme der Mutanten L233C. Die exprimierten Mutanten erzeugten charakteristische Kv-Kanal-ähnliche Ströme. Wie zu erwarten war, führten die untersuchten Mutanten zu signifikanten Verschiebungen des V0.5-Wertes, verglichen mit denen des KCNQ1 Wildtyps. Interessant zu beobachten war, daß Mutanten, die eine Verschiebung des V0.5-Wertes in Richtung negativen Potentials zeigten, sich zwischen G219C und F222C (miteinbezogen auch Positionen G216C und Q234C) befanden. Mutanten die eine positive Verschiebung aufwiesen, waren mehr C-terminal lokalisiert: A223C, T224C, S225C, A226C, G229C, I230C, F232C und I235C. Eine Ausnahme hierzu bildet die Mutante S217C. Eine negative Verschiebung des V0.5-Wertes kann als Stabilisierung des offenen Kanalzustandes beziehungsweise als Destabilisierung des geschlossenen Kanalzustands betrachtet werden, da kleinere Depolarisationen schon ausreichen, um den mutierten Kanal zu aktivieren. Das genaue Gegenteil der obig beschriebenen Situation gilt bei positiven Verschiebungen des V0.5-Wertes, d.h. Stabilisierung des geschlossenen bzw. Destabilisierung des offenen Kanalzustands. Deswegen tendieren Mutationen im N-terminalen Bereich des S4 Segments dazu, den geschlossenen Zustand des Kanals zu destabilisieren. Wohingegen Cystein-Substitutionen weiter abwärts des S4 Segments gelegen vorzugsweise zur Stabilisierung den geschlossenen Kanalzustands führen. Von den 20 untersuchten Mutanten wiesen die Konstrukte A223C bis I227C, I230C, G219C sowie Q234C z.T. drastische Veränderungen nach Inkubation mit MTSES auf, wodurch die extrazelluläre Zugänglichkeit der betreffenden Positionen eindeutig belegt wird. Von den untersuchten Konstrukten, wiesen die Mutanten T224C und S225C in ihrem Verhalten bei depolarisierenden Spannungssprüngen nach MTSES-Inkubation eine Restaktivierung auf. Dagegen zeigten die Ströme, die bei den Mutanten A223C, A226C und I230C aufgenommen wurden, daß diese mit MTSES modifizierten Kanäle bei stark hyperpolarisierenden Potentialen zu einem Deaktivierungszustand getrieben werden können. Die beobachteten Veränderungen in der Stromcharakteristik konnten mit der hyperpolarisierenden Verschiebung des V0.5-Wertes korreliert werden. Dies bedeutet, daß die kovalente Anknüpfung der MTSES-Seitengruppe, das spannungssensitive S4 Segment verhindert, wieder zurück zu einer Kanal-Konformation zu gelangen, welche die Schließung des Kanals ermöglicht. In den meisten Fällen führte die kovalente Anknüpfung der MTS-Seitengruppe zu einem Erscheinungsbild, wie es für konstitutiv offene Kanäle charakteristisch ist. Auch im Falle der Cystein-Insertion an der Spannungssensor-Position R228 waren charakteristische Veränderungen nach MTSES-Zugabe zu beobachten. Die R231C-Mutante (ebenfalls eine der Schaltladungen betreffend), zeigte bereits vor der MTSES-Applikation einen Offenkanal-Phänotyp, infolge MTSES-Zugabe trat keine weitere Veränderung auf. Basierend auf der Kristallstruktur des spannungsgesteuerten Kv1.2-Kanals wurde ein Strukturmodell von KCNQ1 konstruiert, welches dazu diente, potentielle Aminosäure-Positionen innerhalb anderer Transmembrandomänen von KCNQ1 vorzuschlagen, die in unmittelbarer Umgebung der MTSES-zugänglichen S4-Positionen liegen könnten. Dementsprechend wurden Doppelmutanten mit je einem Cystein auf dem S4-Segment und einem auf einer anderen Helix untersucht. Zuvor wurde der KCNQ1-Wildtyp und die einzelnen Cystein-Mutanten auf Cd2+ Sensitivität getestet. Eine Konzentration von 100μM wurde als optimal bestimmt, da keine funktionellen Veränderungen der Kanäle bei dieser Konzentration beobachtet werden konnten. Von den vier vorgeschlagenen Cystein-Paaren konnte für die Kombinationen I230C/M159C und T224C/F279C mittels Cd2+-Quervernetzungsversuchen eine enge Nachbarschaft zwischen den entsprechenden Aminosäure-Positionen gezeigt werden. In beiden Fällen konnte eine Reduktion der steady-state Stromamplituden beobachtet werden, die parallel einherging mit einer entsprechend signifikanten Erniedrigung der Sättigungskurve I/Imax. Beide Beobachtungen sind ein Indiz dafür, daß die obig genannten Doppelcystein-Mutanten sich mit Cd2+ quervernetzen. Diese Quervernetzung führt zu strukturellen Einschränkungen, welche das Öffnen des Kanals verhindern. Die beobachteten funktionellen Konsequenzen der Cd2+-Quervernetzung lassen sich im Sinne einer Stabilisation des Geschlossen-Zustands des Kanals interpretieren. Diese Befunde belegen die Gültigkeit des erstellten Struktur-Modells, welches auf der Kristallstruktur eines geschlossenen Kv-Kanals beruhte
Bayesian oma of offshore rock lighthouses: Surprises with close modes, symmetry and alignment
No full textA set of seven rock lighthouses around the British Isles was studied by a combination of forced and ambient vibration tests executed with some extreme logistical constraints. Forced vibration testing of the circular section masonry towers combined with experimental modal analysis identified modes with alignment assumed the same as the shaker as well as some interesting effects of helideck retrofit, whereas operational modal analysis revealed the considerable degree of uncertainty in mode shape alignment. Hence Bayesian operational modal analysis was used to characterise the uncertainty and find the best representation of mode shape direction. While perfectly axisymmetric towers would show a single frequency omnidirectional mode, OMA reveals the split modes and allows un unbiased view of directionality. The variability and uncertainty of these mode shape directions are further revealed using Bayesian OMA.Accepted Author ManuscriptCoastal Engineerin
COMPARISON OF RAW ACCELERATION FROM CONSUMER WEARABLES AND ACTIGRAPH ACCELEROMETERS USING A MECHANICAL SHAKER TABLE
No full textJames W. White III, Nick Tindall, Olivia Finnegan, Kasey Hansen, Meghan Bastyr, Hannah Parker, Roddrick Dugger, Elizabeth L. Adams, Sarah Burkart, Bridget Armstrong, Michael W. Beets, R. Glenn Weaver. University of South Carolina, Columbia, SC.
BACKGROUND: Though the proprietary signal processing of acceleration output from consumer wearables limits their use for research on physical activity (PA) and sleep assessment, it may be possible to develop open-source prediction equations for estimating PA and sleep based on raw acceleration estimates from these devices. Thus, the aim of this study was to compare raw acceleration output from ActiGraph wGT3X-BT (ActiGraph) and consumer wearables (i.e., Garmin Vivoactive 4S [Garmin] and Apple Watch Series 7 [Apple]) using a mechanical shaker table (Scientific Industries; Mini-300 Orbital Genie, Model 1500). METHODS: A total of 30 devices, including 10 ActiGraph accelerometers and 10 of each consumer wearable were analyzed in this study. Validity of raw acceleration estimates from consumer wearables was tested against a criterion of ActiGraph. Devices were mounted directly to the twin ratcheting clamps of the shaker table and were oscillated at various speeds (i.e., 0.6 Hz, 1.0 Hz, 1.5 Hz, 1.9 Hz, 2.4 Hz, 2.8 Hz, and 3.2 Hz) for 2-minutes each (i.e., 7 speeds for 2 minutes each) until all consumer wearables were compared to all ActiGraph devices. The raw acceleration values for the x, y, and z axes were extracted from the middle minute of each 2-minute speed, and the maximum vector magnitude was calculated for each second. Pearson product moment (r) and Lin’s concordance correlation coefficients (CCC) were calculated. Bland-Altman plots were also constructed with mean bias and 95% limits of agreement. RESULTS: The correlations of Garmin and Apple with Actigraph were r=0.881 and r=0.933, respectively. CCC from raw acceleration estimates for Garmin and Apple were 0.763 and 0.918, respectively. Bland-Altman plots (consumer wearable minus ActiGraph) revealed mean differences 0.044 (95% CI: -0.054, 0.142) between Garmin and ActiGraph and -0.002 (95% CI: -0.097, 0.094) between Apple and ActiGraph. CONCLUSIONS: There was moderate concordance and strong correlation between raw acceleration estimates from Garmin and ActiGraph, while there was strong concordance and correlation between raw acceleration estimates from Apple compared to ActiGraph. Garmin and Apple provide comparable estimates of raw acceleration compared to ActiGraph, suggesting that raw acceleration estimates from consumer wearables can be used to develop open-source prediction equations for estimating PA and sleep. Grant or funding information: Research reported in this abstract was supported by the National Institute of Diabetes and Digestive Kidney Diseases of the National Institutes of Health under Award Number R01DK129215. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health
KCNE1 and KCNE2 provide a checkpoint governing voltage-gated potassium channel α-subunit composition
No full textAbstractVoltage-gated potassium (Kv) currents generated by N-type α-subunit homotetramers inactivate rapidly because an N-terminal ball domain blocks the channel pore after activation. Hence, the inactivation rate of heterotetrameric channels comprising both N-type and non-N-type (delayed rectifier) α-subunits depends upon the number of N-type α-subunits in the complex. As Kv channel inactivation and inactivation recovery rates regulate cellular excitability, the composition and expression of these heterotetrameric complexes are expected to be tightly regulated. In a companion article, we showed that the single transmembrane segment ancillary (β) subunits KCNE1 and KCNE2 suppress currents generated by homomeric Kv1.4, Kv3.3, and Kv3.4 channels, by trapping them early in the secretory pathway. Here, we show that this trapping is prevented by coassembly of the N-type α-subunits with intra-subfamily delayed rectifier α-subunits. Extra-subfamily delayed rectifier α-subunits, regardless of their capacity to interact with KCNE1 and KCNE2, cannot rescue Kv1.4 or Kv3.4 surface expression unless engineered to interact with them using N-terminal A and B domain swapping. The KCNE1/2-enforced checkpoint ensures N-type α-subunits only reach the cell surface as part of intra-subfamily mixed-α complexes, thereby governing channel composition, inactivation rate, and—by extension—cellular excitability
Molecular Movement of S4 and the Related Impedence Zone in Shaker Potassium Channels
No full text Shaker 鉀離子通道是一種電位開關性的離子通道，由四個相同的多肽鏈次單位所組成，其 C 端和 N 端都位在細胞膜內；每個次單位包含六條穿膜蛋白 (S1~S6)，其中S5-S6 形成離子通道的孔洞；在 S4 上因為被發現有規則排列的鹼性胺基酸，因此被認為其和感測電場的變化有關，視為電位感測器。近年對於 S4 運動方式主要有兩種說法：第一種是 S4 利用旋轉並往細胞外位移的方式運動；第二種是 S4 位於通道蛋白質的外圍與細胞膜接觸，利用擺動的方式移動。本論文藉由 S4 上其他非鹼性胺基酸的突變，來探討包圍在S4外側之主要電阻區(即S4運動所必須行經以提供電能變化來開關通道者)之形狀與位置，並期能夠因而更加了解S4去極化時移動的方式與功能作用。在將 S4 上非鹼性胺基酸的位置一一突變成帶正電胺基酸 arginine；我們發現 I364R、L366R突變造成通道的活化速率與不活化速率很明顯變慢，在I364R突變觀察到速率之電壓依賴性顯著增強，L366R突變則否。將 364 和 366 兩個位置再做其他不同性質的突變(K、D、E、A、Q、W)，發現I364位置突變成鹼性胺基酸時對電壓依賴性大致都有明顯增加。而L366位置的鹼性胺基酸突變對活化反應之電壓依賴性的影響都不大，但其不活化反應的電壓依賴性則皆變得極不顯著，而且最終不活化通道之比例皆大幅降低。至於I364及L366這兩個位置的若干突變，則都明顯有活化與去活化速率異常變慢的情形，似乎是大幅提高通道活化反應的能量峰值。從 V367一直到 L375 的非鹼性胺基酸位置突變成arginine記錄不到任何電流，到了S376R 與 H378R突變則又可記錄到電流，且其活化速率之絕對值與電壓依賴性與 WT 相較變化並不大。由 I364R、L366R 突變的結果，推測這兩個位置位在 S4 移動的過程會經過主要電阻區，而I364位置可以使鹼性胺基酸攜帶電荷，L366位置則否，由此可以推測他們所處的環境。綜合上述結果，我們描繪出箝住S4的主要電阻區其位置與形狀：在S4之3條”軸線”，即“R”、“R-1”、“R+1”線上，應分別經過R362(R軸線上)﹔L358、L361及/或I364(“R-1”軸線上)﹔V363與L366之間(“R+1”軸線上)。我們的數據同時也符合鉀離子通道在去極化過程中，其S4 可能移動 9 個胺基酸的距離，並轉動了180 度之想法。Shaker K+ channel is a member of the voltage-gated K+ channels which are composed of four polypeptide subunits. Each of the subunits has six transmembrane segments (S1-S6), with the C- and N-termini both located intracellularly. Because of the regularly spaced basic amino acids, S4 has been considered as the voltage sensor of the channel. S4 presumably can move in response to membrane electric field change and thus cause subsequent gating conformational changes of the channel. There are chiefly two models explaining the S4 movement: the helical model and the paddle model. We made point mutation on S4 unchanged residues in S4, from V363 (which is located right internal to the outermost positive residue R362) to H378. In I364R and L366R mutant channels, the activation and inactivation rates are evidently slower. I364R shows more voltage-dependent gating parameters as compared to the wild type channels, but L366R did not. I364K has similar characteristics to I364R. The other mutations involving I364 (I364D, E, A, Q, W) do not show increased voltage dependence in gating parameters, although the activation and inactivation curve are shifted to different extent in the voltage axis. L366R and L366K not only show no increased voltage dependence in the gating parameters, but also has very slow activation kinetics. Markedly decreased voltage dependence in the inactivation kinetics. Some of the other mutations at L366 (L366D, E, W) also showed slowed gating kinetics. We could not detect any currents in single mutations involving the hydrophobic residues in S4 from V367R to L375R, but S376R and H378R give definite currents. We propose that I364 and L366 move across a significant part of electric field (impedence zone), whereas S376 probably does not cross the field during the gating movement of S4. The impedence zone goes through R362 (in the R axis), L358, L361 and/or I364 (in the “R-1” axis), and between V363 and L366 (in the “R+1” axis). Moreover, our data are also consistent with the proposal that S4 most likely moves through the gating canal by a translation shift of 9 residues and a rotation of ~180°during channel activation.中文摘要……………………………………………………………...….……………3
英文摘要……………………………………………………………………....………5
第一章 導論……………………………………………………………………….....7
第二章 材料與方法……………………………………………………………...…18
第三章 結果……………………………………………………………………...…25
第四章 討論………………………………………………………………………...34
圖次
圖1 電位開關性鉀離子通道topology、S4胺基酸序列與S4模式圖……...….…43
圖2 WT IR、I364R IR、I364K IR之電流記錄與活化曲線………………….…44
圖3 I364突變通道之活化電流記錄與活化曲線………………………….….….45
圖4 WT、I364R、I364K之不活化電流記錄與不活化曲線…………………….46
圖5 I364突變通道之不活化電流記錄與不活化曲線………………………..….47
圖6 I364突變通道之活化速率與電壓依賴性…….………………………….….48
圖7 I364突變通道I-V電流記錄……………………………………………..…..49
圖8 I364突變通道之不活化速率與電壓依賴性…………….………………..….50
圖9 WT IR與I364突變通道之去活化tail current……………………………....51
圖10 不同前置電壓下I364W IR之活化曲線與活化速率……………………....52
圖11 L366R IR與L366K IR之電流記錄與活化曲線…………………………....53
圖12 L366突變通道之電流記錄與活化曲線……………………………….…...54
圖13 L366R、L366K之不活化電流記錄與不活化曲線…………………….….55
圖14 L366突變通道之不活化電流記錄與不活化曲線…….……………….…..56
圖15 L366突變通道之活化速率與其電壓依賴性…….………………….……..57
圖16 L366突變通道I-V電流記錄………………………………………………58
圖17 L366突變通道之不活化速率與電壓依賴性……………………………....59
圖18 L366突變通道之去活化tail current…………….……………………….…60
圖19 L366W、L366W IR、Del 6-9 L366W之活化與不活化曲線……………..61
圖20 V363R活化、不活化曲線，活化、不活化速率及其電壓依賴性……….62
圖21 V363與I360突變通道之活化曲線與活化速率…………………………..63
圖22 S376R、H378R活化、不活化曲線與活化、不活化速率………………..64
圖23 主要電阻區之示意圖……………………………………………………….65
圖24 S4活化反應位移之示意圖…………………………………………………66
參考文獻…………………………………………………………………….……….6
Correction: Vitamin D status and supplementation before and after Bariatric Surgery: Recommendations based on a systematic review and meta‑analysis
No full textThe article “Vitamin D status and supplementation before and after Bariatric Surgery: Recommendations based on a systematic review and meta‐analysis”, written by Andrea Giustina, Luigi di Filippo, Antonio Facciorusso, Robert A. Adler, Neil Binkley, Jens Bollerslev, Roger Bouillon, Felipe F. Casanueva, Giulia Martina Cavestro, Marlene Chakhtoura, Caterina Conte, Lorenzo M. Donini, Peter R. Ebeling, Angelo Fassio, Stefano Frara, Claudia Gagnon, Giovanni Latella, Claudio Marcocci, Jeffrey I. Mechanick, Salvatore Minisola, René Rizzoli, Ferruccio Santini, Joseph L. Shaker, Christopher Sempos, Fabio Massimo Ulivieri, Jyrki K. Virtanen, Nicola Napoli, Anne L. Schafer, John P. Bilezikian, was originally published electronically on the publisher’s internet portal on September 04, 2023 without open access. With the author(s)’ decision to opt for Open Choice the copyright of the article changed on September 09, 2023 to © The Author(s) 2023 and the article is forthwith distributed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0. The original article has been corrected
A comparative study of seeding techniques and three-dimensional matrices for mesenchymal cell attachment
No full textMesenchymal stem cells (MSCs) offer significant potential as a cell source in tissue-engineering applications because of their multipotent ability. The objective of this study was to evaluate the behaviour of MSCs during the seeding phase, using four different seeding techniques (spinner flask, custom vacuum system combined with a perfused bioreactor or with an orbital shaker, and orbital shaker) with four different scaffold materials [polyglycolic acid, poly(lactic acid), calcium phosphate and chitosan–hyaluronic acid]. Scaffolds were selected for their structural and/or chemical similarity with bone or cartilage, and characterized via scanning electron microscopy (SEM) and measurement of fluid retention. Cell attachment was compared between seeding techniques and scaffolds via cell-binding kinetics, cell viability and DNA quantification. SEM was used to evaluate cell distribution throughout the constructs. We discovered from cell suspension kinetics and DNA data that the type of loading (i.e. direct or indirect) mainly influences the delivery of cells to their respective scaffolds, and that dynamic seeding in a spinner flask tended to improve the cellularity of polymer constructs, especially mesh. Regardless of the seeding method, bone marrow-derived MSCs displayed a superior affinity for calcium phosphate scaffolds, which may be related to their hydrophobicity. MSCs tended to aggregate into flat sheets, occluding the external pores of matrices and affecting cell distribution, regardless of seeding technique or scaffold. Taken together, these results provide insight into the design of future experiments using MSCs to engineer functional tissue
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